Inward-rectifier K+ channels function like K+-selective diodes in the cell membrane. They pass much larger inward than outward K+ current under symmetric ionic conditions. This unusual property is commonly referred to as inward rectification, which results from voltage dependent blockade by intracellular cations such as Mg2+ and polyamines. Under physiological conditions, inward rectification manifests itself as a progressive reduction of the outward current, which allows the channel to control and regulate the resting membrane potential without impeding the generation of action potentials. Through regulation of the resting membrane potential inward-rectifier K+ channels accomplish many important and diversified biological tasks. For example, the G-protein-gated K+ channels control the heart rate and modulate neurotransmission; the ATP-sensitive K+ channel couples blood glucose level to insulin secretion; the ROMK1 channel mediates water and electrolyte excretion in the kidney. The activity of most, if not all, inward-rectifier K+ channels are regulated by intracellular signaling pathways such as G-proteins, inositol phosphates and protein kinases.
Inward-rectifier K+ channels differ from voltage-activated K+ channels not only in function but also in structure. Each of the four subunits of the inward-rectifier K+ channels has only two transmembrane segments rather than six found in voltage-activated K+ channels. The amino acid sequences between the two channel types are minimally conserved except for the signature sequence that forms the K+ selective filter. Although most of the inward-rectifier K+ channels are formed by four identical subunits, some channels are formed by non-identical subunits. An example of a non-identical subunit is the G-protein gated inward-rectifier K+ channel (GIRK1/4) in the heart, which is formed by two different types of subunits, GIRK1 (GSK) and GIRK4 (CIR). In some cases, the channels are complexed with other proteins. For example, the ATP-sensitive K+ channel is a complex of an inward-rectifier K+ channel (Kir 6.2) and sulfonylurea receptor.
It has been well established that scorpion toxins inhibit the voltage- and Ca2+-activated K+ channels by blocking the ion conduction pore (MacKinnon, R. and Miller, C. J. Gen. Physiol. 1988 27:8491–8698; Miller, C. Neuron J. 1988 1:1003–1006; Park, C.-S. and Miller, C. Neuron 1992 9:307–313). Extensive mutagenesis studies have revealed much of the molecular interactions between the toxins and the channels. Recent crystallographic studies on a bacterial K+ channel showed how the P-region makes up the outer part of the pore. The signature sequence forms the K+-selective pore and the residues C-terminal to the signature sequence form the base of the external vestibule. The sequence N-terminal to P-region produces four turrets that surround the pore. When a scorpion toxin blocks the channel, it lies between two diagonally located turrets. The middle portion of the toxin contacts the vestibule base while the two ends contact the turrets. Because the channel is four-fold symmetric, a toxin molecule can bind to the channel in four equivalent orientations.
However, the pharmacology of inward-rectifier K+ channels is not well developed. No high affinity ligands that directly target any inward-rectifier K+ channels have been identified in the prior art. Out of the various scorpion toxins that target K+ channels, only Lq2 and Δ-dendrotoxin block the ROMK1 inward-rectifier K+ channel and the affinities are rather low (Kd=0.4 and 0.15 μM, respectively) (Lu, Z. and MacKinnon, R. Biochemistry 1997 36:6936–6940; Imredy et al. Biochemistry 1998 37:14867–14874).
Accordingly, there is a need for high affinity inhibitors against inward-rectifier K+ channels.
Tertiapin is a small protein in honey bee venom which was initially purified over 20 years ago (Gauldie et al. Eur. Biochem. 61, 369–376). Because the venom was believed to contain materials beneficial to arthritis, many laboratories tried to identify the anti-arthritic components in the venom. This search led to the purification of many small proteins. Two of the purified small proteins, apamin and mast cell degranulating peptide (MCDP), were found to be inhibitors of voltage- and Ca2+-activated K+ channels (Blatz, A. L. and Magleby, K. L. Nature, 1986 323:718–720; and Stuhmer et al. EMBO J. 1989 8:3235–3244). However, tertiapin was one of the many other purified proteins without any clearly identified biological activity.
While the biological activity of tertiapin was unknown, the studies on tertiapin chemistry were quite advanced. The three-dimensional structure of tertiapin has been determined using NMR spectroscopy (Xu, X. and Nelson, J. W. Protein: Structure, Function and Genetics 1993 17:124–137). The structure shows that tertiapin is a highly compact molecule with a high density of positively charged residues. It consists of a type 4 reverse turn and an α-helix. A loop formed by an extended β sheet connects the turn and the helix. Four cysteines within the polypeptide chain form two disulfide bonds. The extensive interactions among the side-chains enhance the rigidity of the structure of tertiapin. The overall structure of tertiapin is very similar to that of apamin (Pease, J. H. and Wemmer, D. E. Biochemistry 1988 27:8491–8498). The main difference between these two structures is the relative position of the connecting loop and the α-helix. This difference is caused by the existence of an extra amino acid residue in the connecting loop of tertiapin.
Tertiapin has now been purified and identified as an inhibitor against two members of the inward-rectifier K+ channel family. Both the GIRK1/4 and ROMK1 inward-rectifier K+ channels are highly sensitive to tertiapin. Based upon homology, it is expected that ATP-sensitive K+ channels will also be sensitive to tertiapin.